Biogenesis of RNase P RNA from an intron requires co-assembly with cognate protein subunits

Abstract RNase P RNA (RPR), the catalytic subunit of the essential RNase P ribonucleoprotein, removes the 5′ leader from precursor tRNAs. The ancestral eukaryotic RPR is a Pol III transcript generated with mature termini. In the branch of the arthropod lineage that led to the insects and crustaceans, however, a new allele arose in which RPR is embedded in an intron of a Pol II transcript and requires processing from intron sequences for maturation. We demonstrate here that the Drosophila intronic-RPR precursor is trimmed to the mature form by the ubiquitous nuclease Rat1/Xrn2 (5′) and the RNA exosome (3′). Processing is regulated by a subset of RNase P proteins (Rpps) that protects the nascent RPR from degradation, the typical fate of excised introns. Our results indicate that the biogenesis of RPR in vivo entails interaction of Rpps with the nascent RNA to form the RNase P holoenzyme and suggests that a new pathway arose in arthropods by coopting ancient mechanisms common to processing of other noncoding RNAs.

iv containing the RFP exons, split by D. virilis intron-containing RPR. The primer sequences to generate the three fragments are as follows (see Supplementary   Table S5 for sequence details): Fragment 1: 'EcoRV forward primer' and Dv RPR 5T-mutations reverse primer; Fragment 2: 'Dv RPR 5T-mutations forward primer' and 'Dv RPR 4T-mutation reverse primer'; Fragment 3: 'Dv RPR 4T-mutation forward primer' and 'XbaI reverse primer'. A single fragment (1.5 Kb) containing the RFP exons, split by the D. virilis intron-RPR containing polyT mutations, was generated using SOE-PCR that spliced together three DNA fragments using the EcoRV forward primer and the XbaI reverse primer (forward primer used for 'Fragment 1' and reverse primer used for 'Fragment 3', respectively (see Supplementary Table S5)). This fragment was cloned using recombination (InFusion cloning kit) into pPacPL that had been digested with EcoRV and XbaI to excise the RFP exons and wild type D. virilis intron.
6. pMRP promoter-Dv RPR 5T4T (Supplementary Figure S5D): A DNA fragment corresponding to the D. virilis RPR (with the 5T4T mutations) under the control of the D. melanogaster MRP promoter was generated by GENEWIZ (see Supplementary Table S4 for sequence details). This fragment was cloned using recombination (InFusion cloning kit) into the linearized pBlueScript (KS-) vector that had been digested with EcoRI and BamHI. (C) RT-PCR to detect Ldbr knockdown (KD) efficiency using as input RNA extracted from S2 cells that were first treated with dsRNA to knockdown Ldbr or GFP and subsequently transfected with the split-RFP reporter. GAPDH was used as the loading control for RT-PCR (Related to Figure 1A and 1B).
(D) Histogram and accompanying table indicate raw splice-site scores for the 5′ donor and 3′ acceptor splice-sites (ss) for the first and second (RPR-containing) intron of the ATPSynC gene in 12 annotated Drosophila species. Zero indicates that a splice-site score was not predicted for that exon-intron junction (Related to Figure 1D).
vi (See Supplementary Table S1 for sequences of probes used, and Supplementary Tables   S2 and S3 for  (B) RT-PCR to determine the expression level of Rat1, Dis3, or Rrp6 for RNA extracted from S2 cells that were treated with dsRNA to either the specified nuclease or GFP and subsequently transfected with the split-RFP reporter. PCR products were quantitated by ImageJ and normalized to GAPDH (data related to northern blots in Figures 2A and   2G).
(C) RT-PCR to determine the expression level of Rat1 for RNA extracted from S2 cells treated with dsRNA to knockdown either Rat1 or GFP and subsequently transfected with the split-RFP reporter containing non-functional splice-sites. PCR products were quantitated by ImageJ and normalized to GAPDH. These RT-PCR data relate to the knockdown experiment depicted in Figure 2B.
vii (D-F, top) Northern blot of RNA extracted from S2 cells that were first treated with dsRNA to knockdown the indicated nuclease (Rex2, IntS11 or Rexo5) or GFP and subsequently transfected with the split-RFP reporter (Supplementary Figure S2A). Hybridization with an antisense-RPR probe (Probe 1) showed that the RPR levels were unaffected. RT-PCR indicating knockdown of Rai1; these data relate to the knockdown experiment depicted in Figure 2A. (See Supplementary Table S1 for sequence of probe1 used for northern blot detection) (D-F, bottom) Efficiency of knockdown for a given nuclease as determined by RT-PCR and compared to GFP. GAPDH was used as the loading control.
(G) Efficiency of knockdown for Ldbr, Rat1 or GFP as determined by RT-qPCR. GAPDH was used as the normalization control. These data relate to the knockdown experiment in Figure 2F.    xvi Legends for supplementary tables Supplementary Table S1. DNA probes used for northern hybridization. The probes are shown graphically in Figure   2A (probes 1-3) and Supplementary Figure S2A (probes 1-7).

Supplementary Table S2.
Primers used to generate gene-specific PCR products with T7 promoters used as templates for in vitro transcription (IVT) of dsRNAs for RNAi experiments.
PCR products for Rai1 and Rex2 were obtained directly from the DRSC (Drosophila RNAi Screening Center. There are two Rpp14 genes in Drosophila, CG34317 (*) and CG15526.
A dsDNA template was generated to target CG34317, which is expressed in S2 cells.

Supplementary Table S3.
Primer sequences used for PCR and qPCR to detect knockdown efficiency for the indicated gene.

Supplementary Table S4.
DNA sequence for the RFP exons, split by D. virilis intron-RPR and DNA fragments synthesized by Genewiz.

Supplementary Table S5.
Oligonucleotides used for construction of plasmids and reporter genes used in this study.  Table S1. DNA probes used for northern analysis (Figures 1-3 Table S2. Primers to create T7-dsDNA template for generation of dsRNA used for RNAi mediated knockdowns, related to main Figures 1-3, S1-S4